Granuloma pouch assay for mutagenicity testing

The Granuloma Pouch Assay (GPA) is an animal model in which mutagenic and carcinogenic effects of a testcompound can be detected in rapidly dividing fibroblasts of a granulation tissue in adult male rats. Growth of this tissue was initiated with a small amount of croton oil at the inside wall of a subcutaneous air pouch on the back of the animals. The test compound can be injected either into the pouch (local) or administered by systemic routes. Alkali labile DNA-lesions, chromosome aberrations, sister chromatid exchanges, point mutations and tumor development in situ were determined. The comparison of mutation frequencies after local and systemic administration of testcompounds, provide an estimation of the pharmacokinetic characteristics and the mutagenic potency of the chemical. The local application route allows the detection of locally active mutagens and of compounds which require activation by P-448 dependent monooxygenases. Liver mediated proximate metabolites are detectable when they are transformed into ultimate carcinogens in extrahepatic cells whereas chemicals with a strong organ specific activity are not.     

Covalent binding of drug metabolites to DNA — a tool of predictive value?

The presently available data suggest at least some correlation between covalent binding of drug metabolites to DNA and carcinogenicity of that drug. More data, however, are needed to establish the predictability of covalent DNA binding assays for extrahepatic cancer. A covalent binding assay requires administration of radioactively labelled compound to the experimental animals; the availability of labelled compound and requirements as to radiochemical purity, chemical and biochemical stability are limiting the applicability of this procedure. Many technical pitfalls accompany covalent DNA binding assays. It is concluded that at the present time DNA binding assays do not represent routine procedures within a standard test battery for carcinogenicity, but are invaluable for more in-depth research which probably follows routine testing.     

International programme for the evaluation of short-term tests for carcinogenicity

A brief report of the design and initial results from the International Programme for the Evaluation of Short-Term Tests for Carcinogenicity is presented. A total of 42 chemicals, including 14 structurally-related, carcinogen/non-carcinogen pairs were coded and submitted for testing in 35 assays. Several of the assays provided good predictions of carcinogenic potential. The results revealed the advantages and disadvantages of the assays used and provide some indication of the applicability of the tests to screening for carcinogenicity.To be published by Elsevier, North Holland under the title Short-Term Tests for Carcinogens; Report of the International Collaborative Program.     

刮除术配合三氧化铁治疗化脓性豆芽肿临床研究

目的为了寻找一个有效、安全、简单的方法治愈化脓性肉芽肿。方法采用刮除术配合三氯化铁治疗化脓性肉芽肿。结果自2003年1月至2007年9月确诊为化脓性肉芽肿患者98例，随机分成两组：治疗组52例，对照组46例。治疗组52例治愈率100％，肿物完全消失，3个月内无复发。结论采用刮除术配合30％三氯化铁止血治疗化脓性肉芽肿是一个高效、安全、简单的治疗方法，无不良反应，利于临床推广。     

Phenolphthalein and bisacodyl: assessment of genotoxic and carcinogenic responses in heterozygous p53 (+/-) mice and syrian hamster embryo (SHE) assay.

Phenolphthalein (800 and 2400 mg/kg/day by gavage and 2400 mg/kg/day by diet) and bisacodyl (800-500, 4000-2000, and 8000 mg/kg/day by gavage) were administered to 15 male and 15 female and 20 male and 20 female p53(+/-) mice respectively for 26 weeks to investigate the potential carcinogenicity of each compound. Toxicokinetic analyses confirmed systemic exposure. p-Cresidine was administered by gavage (400 mg/kg/day) and served as the positive control agent in each study. Dietary phenolphthalein reduced survival in both sexes and early deaths were attributed to thymic lymphoma. No bisacodyl-related neoplasms were observed. Regardless of route of administration to p53(+/-) mice, phenolphthalein but not bisacodyl was unequivocally genotoxic, causing increased micronuclei in polychromatic erythrocytes. In the Syrian hamster embryo (SHE) cell transformation assay, phenolphthalein caused increases in morphologically transformed colonies, thereby corroborating NTP's earlier reports, showing phenolophthalein has potential carcinogenic activity. Bisacodyl was negative in the SHE assay. Results of these experiments confirm an earlier demonstration that dietary phenolphthalein causes thymic lymphoma in p53(+/-) mice and show that (1) phenolphthalein causes qualitatively identical results in this transgenic model regardless of route of oral administration, (2) phenolphthalein shows evidence of micronucleus induction in p53(+/-) mice for up to 26 weeks, (3) phenolphthalein induced transformations in the in vitro SHE assay, and (4) bisacodyl in p53(+/-) mice induces neither drug-related neoplasm, nor micronuclei in polychromatic erythrocytes, and did not induce transformations in the in vitro SHE assay.     

Acceleration phenomenon of granuloma formation caused by felt-pellet in rats

Acceleration phenomenon of granuloma formation was studied using the technique of feld-pellet implantation in rats. When the 2nd felt-pellets were implanted into the back of the same rat 3 or 6 days following the 1st implantation, the dry weight (93.4 mg) of granulation tissue formed around the 2nd implanted pellet was significantly heavier than that in the one-time pellet implantation (69.0 mg). The result demonstrated that the growth of granulation tissue induced by the 2nd pellet was accelerated by the 1st pellet implantation. However, the acceleration phenomenon was not observed when the 2nd pellet was implanted on the 14th day following the 1st pellet implantation, or in adrenalectomized rats. The acceleration of granuloma formation was not observed in the rat with the inflammation such as yest-induced granuloma pouch or adjuvant-induced hind paw oedema. The mode of action of the acceleration phenomenon is discussed.     

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.     

连续间置空肠在全胃切除术中的应用

目的探讨全胃切除术后理想的重建方式。方法对62例连续间置空肠代胃术及60例Roux-en-Y重建术后1年随访资料进行对比。结果连续间置空肠（CJIP）组每日进餐次数较后者少（P〈0．05），每餐进食量及代胃肠段最大直径较后者大（P〈0．05）；且后者有倾倒症状8例，胆汁反流9例。但反映营养状况的体质量变化、血红蛋白及总蛋白、白蛋白量两组差异无统计学意义（P〉0．05）。结论连续间置空肠法营养状况的改善虽然并不优于Roux-en-Y法，但术后不适少于后者，因此适宜的患者应优选间置空肠法重建消化道。     

运用小鼠淋巴瘤细胞基因突变试验测定4,4′-二甲基二苯基碘鎓盐六氟磷酸盐的遗传毒性

目的研究运用小鼠淋巴瘤试验(MLA)对4,4′-二甲基二苯基碘鎓盐六氟磷酸盐(IHT-PI820)作为食品外包装上油墨使用安全性的评价。方法采用L5178Y细胞,处理3h,表达48h后测定突变细胞频率及小集落比例。同时进行添加和未添加代谢活化系统的试验,每个条件下设6个剂量组,阴性(二甲亚砜)、阳性(甲磺酸甲酯或环磷酰胺)对照组及IHT-PI8204个剂量组(37.5,75.0,150.0和300.0mg·L-1),在相同条件下,每组设定两个平行样。结果IHT-PI820各剂量组和阳性对照组的突变频率均显著性高于阴性对照组,并且IHT-PI820各剂量组间存在明确的剂量反应关系,小集落的比例也随剂量的升高呈上升趋势。结论IHT-PI820可以诱发小鼠淋巴瘤细胞Tk基因突变,故将其作为食品外包装上的油墨使用时,应严格按照相关的行业规定操作。     

运用小鼠淋巴瘤细胞基因突变试验测定4,4′-二甲基二苯基

目的研究运用小鼠淋巴瘤试验(MLA)对4,4′-二甲基二苯基碘鎓盐六氟磷酸盐(IHT-PI820)作为食品外包装上油墨使用安全性的评价。方法采用L5178Y细胞,处理3h,表达48h后测定突变细胞频率及小集落比例。同时进行添加和未添加代谢活化系统的试验,每个条件下设6个剂量组,阴性(二甲亚砜)、阳性(甲磺酸甲酯或环磷酰胺)对照组及IHT-PI8204个剂量组(37.5,75.0,150.0和300.0mg·L-1),在相同条件下,每组设定两个平行样。结果IHT-PI820各剂量组和阳性对照组的突变频率均显著性高于阴性对照组,并且IHT-PI820各剂量组间存在明确的剂量反应关系,小集落的比例也随剂量的升高呈上升趋势。结论IHT-PI820可以诱发小鼠淋巴瘤细胞Tk基因突变,故将其作为食品外包装上的油墨使用时,应严格按照相关的行业规定操作。     

预置膀胱颈部荷包缝合在耻骨上经膀胱前列腺摘除术中应用(附15例报告)

目的通过预置膀胱颈部荷包缝合在耻骨上经膀胱前列腺摘除中应用,探讨更简便,有效的止血方法.方法对15例前列腺增生症患者采用预置膀胱颈部荷包缝合耻骨上经膀胱前列腺摘除术,使用1号普通羊肠线做膀胱颈部荷包缝合,摘除腺体后,对膀胱颈部不再进行其它缝合止血处理.与同期传统耻骨上经膀胱前列腺摘除术16例患者进行比较.结果采用预置膀胱颈部荷包缝合方法,在手术时间、输血量、术后尿管牵拉时间均少于传统的手术方法.术后随访1～4年,无尿道狭窄,膀胱结石发生.结论预置膀胱颈部荷包缝合在耻骨上经膀胱前列腺摘除术中应用,是一种简便、有效的手术止血方法.     

Benefit of antibiotic therapy on pouchitis after ileal pouch anal anastomosis: A systematic review and meta-analysis of clinical trials

The aim of the study was to evaluate and collect current evidence on the effect of antibiotics in pretreatment of pouchitis after restorative ileal pouch anal anastomosis (IPAA). Pubmed, Embase, Web of Science, Scopus, and Cochrane Library databases were searched between 1966 and July 2008; and relevant clinical trials extracted, reviewed, and validated according to the study protocol. The outcome of interest was clinical improvement after treatment. Nine randomized, placebo-controlled clinical trials were found relevant and studied but 3 of them with 70 patients were entered into meta-analysis. Pooling of the results from these trials yielded an odds ratio of 15.96 with a 95% CI of 4.20–60.70, indicating a significant OR (p<0.0001) in treatment group in comparison to the placebo group. In conclusion, the meta-analysis confirms benefit of antibiotics in management of pouchitis after IPAA operation.     

结肠成形术预防低位直肠癌术后前切除综合征的疗效分析

目的评价低位直肠癌前切除术中采用结肠成形术（transverse coloplasty pouch anastomosis）对预防前切除术后综合征的疗效。方法将2007年8月至2008年7月行低位直肠癌前切除术的直肠癌患者40例随机分为两组,一组采用结肠成形术20例,制作结肠贮袋长约5 cm,另一组采用传统的结肠、直肠端端吻合术术（end to end anastomosis）20例,通过术后3,6,9,12个月随访比较两组手术并发症及排便功能。结果术后并发症中,两组均无死亡病例、无临时性造口病例,无吻合口漏发生,无吻合口狭窄发生。直吻组出现1例吻合口出血,2例局部复发,成形组1例局部复发。术后6个月时排便功能成形术组优于传统组,差异有统计学意义（P〈0.05）;术后9个月时两组各项指标比较,仅大便失禁评分成形组优于传统组,差异有显著性（P〈0.05）,其他各项指标比较均无明显差异（P＆gt;0.05）;术后12个月以上两组上述指标基本类似,两组比较,差异无统计学意义（P〉0.05）,结论对于低位直肠癌在行根治性手术的前提下采用结肠成形术可以明显改善术后一年左右的直肠功能,预防术后前切除术后综合征并且不增加术后并发症。     

MTT法检测rhEGF生物活性

目的鉴于待测的生物活性因子均有适于其自身的最佳测定条件,规范了重组表皮生长因子(rhEGF)的MTT测定法。方法根据MTT测定法原理,在3个因素(每孔细胞数、MTT和胎牛血清浓度)及3个水平上进行了正交实验设计。结果MTT检测法的最佳条件是:Balb/c3T3细胞悬液(5×107cells·L-1)100μl·well-1,MTT(8g·L-1)20μl·well-1,FBS(1·5%)100μl·well-1。结论上述正交实验规范的MTT最佳实验条件,适用于Balb/c3T3细胞活性的检测及其药物抑制性实验。但在测定rhEGF生物活性时,为使对照孔细胞数保持在较低水平以便给生物活性因子留有适当的余地以显示其促细胞增殖活性,建议FBS浓度采用0·5%。     

Micronization enhances the protective effect of purified flavonoid fraction against postischaemic microvascular injury in the hamster cheek pouch

1. The present study was designed to evaluate the effect of micronization on the protective effect of the purified flavonoid fraction (MPFF) on increases in macromolecular permeability induced by ischaemia-reperfusion in the hamster cheek pouch microcirculation. 2. Male hamsters (Mesocricetus auratus) were treated orally, twice a day, with vehicle (lactose), MPFF and non-micronized purified flavonoid fraction (PFF) at 5, 20, 80 and 320 mg/kg per day for 10 consecutive days. On the 11th day, cheek pouches of anaesthetized animals were prepared for intravital microscopy. 3. Local ischaemia was obtained by clamping the neck of the everted pouch and the increase in microvascular permeability was quantified as leakage (leaks) of intravenously injected fluorescein isothiocyanate-labelled dextran (FITC-dextran 150; MW = 150 000). 4. Reperfusion, after 30 min ischaemia, resulted in an immediate but reversible increase in post-capillary leakage. The MPFF induced a significant dose-related reduction in the increased permeability, with 83.4% inhibition compared with control at 320 mg/kg per day (19.2 +/- 1.9 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001). Non-micronized PFF was significantly less effective: only 47.9% inhibition compared with control was observed at 320 mg/kg per day (60.3 +/- 1.0 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001) and there was no dose-effect relationship. 5. In conclusion, micronization significantly enhances the protective effects of the purified flavonoid fraction on reperfusion injury in the hamster cheek pouch. This improvement is likely to be related to the better absorption of the micronized formulation, which could explain the superior clinical efficacy shown in previous studies.     

Contribution of toxicology towards risk assessment of carcinogens

In the last decade many tests have been designed to detect possible carcinogenicity of compounds. Presently, many more or less simple and convenient systems are available to detect mutations, effects on chromosomes, DNA binding and damage and malignant transformation. These systems, which have been extensively refined during the last years, often show reasonably good relevance to carcinogenicity. Although inconsistencies in the patterns of response do indicate that their role as predictive indicators of carcinogenicity remains still uncertain, the use of such short-term tests in carcinogen risk assessment does seem feasible.Factors other than these tests should also be taken into consideration, since other characteristics like chemical structure, biotransformation, toxicokinetics, qualitative and quantitative physiological and/or morphological effects, species, strains, organ specificity, dose-response relation and information on studies in man, if available, are of importance too.In conjunction with the results of adequately performed carcinogenicity tests in mammals, one may attempt to classify carcinogens. Current knowledge does not permit a rigid classification, but may warrant a subclassification into carcinogens acting via a genetic or a non-genetic mechanism. It is emphasized that on theoretical and practical grounds a different extrapolation system should be used for the different types of carcinogens in risk assessment procedures. Evaluations on individual compounds should be made to decide whether such genotoxic or non-genotoxic compounds should be permitted in the human environment.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Appreciation of the value of different bacterial test systems for detecting and for ranking chemical mutagens

In more than thirty years of intensive research and development in the field of bacterial mutagenicity testing, a wide range of strains with various genetic endpoints has become available for routine studies. Selective genetic systems of great simplicity and handiness have been developed which assay for gene mutations, such as reversions from auxotrophy and forward mutations to antimetabolite resistance, lysogenic induction of prophages, and DNA repair. The introduction of techniques which take the mammalian metabolism into account, furthermore, makes bacterial systems potentially useful in (i) the primary identification of mutagens and non-mutagens and (ii) the quantification and ranking of relative mutagenic potency.For primary identification purposes, sensitive strains of Salmonella typhimurium LT2 and of Escherichia coli B and K-12 with altered DNA repair capacities have been constructed which demonstrate a high predictive value for many chemical mutagens (and carcinogens). Comparitive studies showed that techniques using these strains can be efficiently standardized and calibrated. Several classes of chemicals with known mutagenic properties, however, are still not detected, or underestimated, in using standard assay procedures. Examples are presented of such compounds, which can be efficiently detected as mutagens upon — slight — modifications of the experimental conditions or the DNA repair capacity of the tester strains. It appears, therefore, advisable to modify and adjust the standard testing protocol to the particular type of chemical under study and to thoroughly calibrate the system with appropriate mutagenic and non-mutagenic reference compounds.For the quantification of mutagenic potency, question remains whether bacterial systems will be of general usefulness. There are indications that the mutagenic activity of certain classes of chemicals, e.g., oxazaphosphorines, aflatoxins, nitroimidazoles, shows some proportionality with their chemical reactivity and that the relative degree of mutagenicity is similar in bacteria and in eukaryotic systems. The attractive possibility of employing in the quantification process those tester strains used for the primary identification appears, however, problematic, because (i) back mutation systems are rather specific in their response to certain mutagens and (ii) the use of strains with altered DNA repair and metabolism may lead to gross overestimations or underestimations of mutagenic potency in the corresponding wild types. More systematic studies are needed to determine the most representative genetic endpoints and genetic background under accurate determination of dose to the target molecule. The feasibility of such quantitative comparative studies has recently been demonstrated in a variety of organisms including bacteria, fungi, Drosophila, and mammalian cells in vitro and in vivo. Proposals are made to use in further comparative studies chemicals, such as procarbazine, cyclophosphamide, and ethylnitrosourea, for which data on gene mutation induction in germinal cells of mammals are available.     

连续监测法测定血清中胆碱酯酶

采用连续监测法测定血清中的胆碱酯酶,其线性相关 r=0.9981,Y_(CHE)=1.08X—7.6,精密度实验批内 CV=3.05%,批间 CV=3.66%,平均回收率为103.0%。11.0mmol/LGLV,42umol/LTBIL 和0.5g/Hb 对测定结果干扰,用本法测定40例正常成人血清(男/女23/17)其=589ou/L 参考值范围(±1.96S)为3864—8096μ/L,本法简单快速,结果准确可靠,适合于日常临床生化检验工作中应用。     

血清脂肪酶乳浊度速率分析法

我们应用乳浊度速率分析法测定血清脂肪酶,线性相关V=0.9933,Y_(LPS)=2119_A-8.6,平均回收率为101.3％,精密度试验批内CV为6.1％,批间CV为8.0％。TBlL80umol/L,TG2.16mmol/L,GLV10.5mmol/L对结果无干扰。用本法测定42名正常健康成人,其参考值为12.4-184V/L(±1.96S)。本方法准确,可靠,并具有简单快速的特点,可做为临床化学分析的常规检测。     

A comparative study of the anticlastogenic effects of chlorophyllin on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz (α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo

Chlorophyllin (CHL), a water soluble derivative of chlorophyll has been shown to have both anticarcinogenic and antigenotoxic properties. We evaluated the protective effects of CHL (25 μM in vitro, 4 and 100 mg/kg. b.w.) on the clastogenic action of two model carcinogens, MNNG and DMBA (25 μM and 2 μM respectively) in vitro on human hepatoma cells (HepG2) and (40 mg and 25 mg/Kg/b.w. respectively) in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with CHL and the carcinogen were carried out in vitro. With MNNG, only simultaneous treatment with CHL was effective in reducing the frequencies of MN, suggesting a direct interaction between CHL and MNNG. A statistically significant reduction in of DMBA induced MN was found by pre-or post treatment with CHL while a reduction (not significant) was observed by simultaneous treatment. In in vivo experiments, CHL pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by MNNG or DMBA. However, increased the toxic effect of DMBA (reduction in percent of PCEs) was accompanied by a reduction in the induced frequencies of MN. CHL was not clastogenic in both in vitro and in vivo tests. It can be concluded that (a) CHL has a protective effect against MNNG and DMBA. This effect is dependent upon the protocol employed in in vitro experiments. In vivo, CHL did not have a protective effect against MNNG and DMBA. A protective effect of CHL against DMBA was evident only at high toxic levels.